Molecular and Biochemical Parasitology. 50 (1992) 151 160 ~;~ 1992 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/92/$05.00

15 l

MOLBIO 01650

Loss of the GP46/M-2 surface membrane glycoprotein gene family in the Leishmania braziliensis complex D i a n e M c M a h o n - P r a t t 1, Y a r a T r a u b - C s e k o 1"*, K e n t o n L. L o h m a n l'**, D . D . R o g e r s 2 a n d S t e p h e n M. Beverley 2 I Yale University, Department of Epidemiology and Public' Health, New Haven, CT, U.S.A. and aDepartment of Biochemistry and Molecular Pharmacology, Harvard School of Medicine, Boston, MA, U.S.A. (Received l l February 1991; accepted 16 August 1991)

Immunization with the GP46/M-2 membrane glycoprotein of Leishmania amazonensis has been shown to induce a protective immune response against infection. We have surveyed a variety of trypanosomatid species and genera for the presence and expression of this gene family, information that will be relevant to future vaccine studies against leishmaniasis. Molecular karyotype analysis revealed the presence of GP46/M-2 genes in all members of the Leishmania mexicana complex, Leishmania major, Leishmania donovani, Leishmania tarentolae, and Crithidia.fasciculata. In contrast, DNAs from species of the Leishmania braziliensis complex (L. braziliensis, Leishmania guyanensis, and Leishmania panamensis) failed to hybridize to GP46/M-2 probes. Western blot analyses with several polyclonal antisera against the GP46/M-2 protein revealed protein expression in L. major and L. donovani, but not L. panamensis or L. braziliensis. Phylogenetic analysis suggests that a loss of the GP46A gene family occurred following separation of the L. braziliensis complex, prior to speciation events within this complex. These data indicate that GP46/M-2 membrane glycoprotein may not be critical to parasite survival, but may play an ancillary role during the developmental cycle. Key words: Trypanosome; Molecular karyotype; Protective antigen; Protozoon

Introduction

The GP46/M-2 membrane protein of Leishmania amazonensis is a major surface component of the promastigote (insect vector) developmental stage of the parasite [1 3], constituting approximately 1-2% of the total membrane protein. The GP46/M-2 protein has Correspondence address." D. McMahon-Pratt, Department of Epidemiology and Public Health, Yale University School of Medicine, P.O. Box 3333, 60 College Street, New Haven, CT 06510, U.S.A. Present addresses." *Fundacao Oswaldo Cruz, Department of Molecular Biology, Rio de Janeiro, R.J., Brazil. **Southwest Foundation for Biomedical Research. San Antonio, TX 78284. U.S.A. Abbreviations: FBS, fetal bovine serum: SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis.

been demonstrated, in conjunction with certain adjuvants, to elicit protective immunity against infection caused by L. amazonensis [4]. Structural studies indicate that the GP46/M-2 molecule is a glycoprotein molecule [3] which is attached to the surface membrane via a phosphatidylinositol-linked lipid anchor rather than protectively embedded within the lipid bilayer [5]. Neither the lipid anchor nor associated carbohydrate side chain appear to be responsible for the overall stability of the molecule [5]. Based on the amino terminal sequence of the GP46/M-2 protein, the gene encoding the protein has been cloned and sequenced from L. amazonensis [6]. The D N A sequence data are consistent with the known biochemical and immunochemical features of the molecule and predicts a repetitive sequence (24 amino acids repeated 4 times) within the amino terminal portion of the molecule which

152

constitutes approximately 22% of the total mature protein: the carboxy-terminal domain consists of proline-rich and cysteine-rich areas of sequence, which likely accounts for the stability of this portion of the molecule to proteolytic digestion. The sequence of the molecules, however, is unique and appears not to be related to any other molecule sequenced to date. We were interested to determine the phylogenetic distribution of the GP46/M-2 gene as well as the expression of the protein within various members of the genus. This information is relevant for future vaccine studies against leishmaniasis and potentially might provide insight to the function of the molecule. Current data suggest that the GP46/M-2 gene family of L. amazonensis is heterogeneous and encodes a family of nonidentical proteins (refs. 6 and 7; P.J. Langer, personal communication). The studies detailed in this paper show that members of the GP46/M-2 family occur and are expressed in species of the Leis'hmania major, Leishmania donovani and Leishmania mexicana complexes, but not in the Leishmania braziliensis complex. Evolutionary considerations suggest that the gene(s) may have been deleted in the L. braziliensis complex.

Materials and Methods

Parasite stocks and culture. The parasites used in this study and the sources of original stocks are listed in Table I. Organisms were cultured in either Schneider's Drosophila medium or Medium 199 containing 10 to 20% heat-inactivated fetal bovine serum (FBS; Gibco or Hyclone) according to methods described [8,9]. Molecular karyotype analyses. Leishmania chromosomes were prepared in agarose plugs and stored at 4°C in 200 mM Tris/100 mM EDTA, pH 8.0 (storage buffer) as described [10]. Pulsed field gel electrophoresis (PFGE) was performed using the contour-clamped homogeneous electric field (CHEF) method

o f C h u et al. [11], using an apparatus similar to that described by these authors or a Bio-Rad Model C H E F - D R II. Electrophoresis was performed in 0.5 x TBE buffer (45 mM Tris/45 mM boric acid/l mM EDTA, pH 8.3) at 4°C using a voltage gradient of 6 V cm ~ at a pulse time of 70 s for 24 h followed by a pulse time of 150 s for 24 h. The gels were blotted to Gene-Screen Plus (Dupont) or Nytran (Schleicher and Schuell), and hybridized with radiolabelled probes as described [12], using a hybridization temperature of 6 0 C in 2 x SSPE (1 × SSPE is 130 mM NaCI/10 mM NaPO4/I mM EDTA, pH 7.6). Post-hybridization washes were 2 x 15 min with 2 × SSPE, 60'~C, 2 x 15 min with 2 x SSPE, 0.5% S D S a t 6 0 ~ C , a n d 2 x 5 m i n w i t h 0 . 1 x SSPE at room temperature. Previous studies with a variety of hybridization probes have shown that these conditions yield specific hybridization among divergent sequences, while further decreases in temperature yield extensive nonspecific hybridization (unpublished data). Size markers were oligomers of lambda DNA (Bethesda Research Laboratories). Restriction enzyme digestion of chromosomes in agarose plugs was performed as described [12]. Probes used for hybridization are indicated in the figure legends [6]. Gel electrophoresis and Western blot analyses. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDSPAGE), was performed using 10% polyacrylamide without prior reduction of the samples [14]. Western blot analyses were performed as previously described [3] according to the methods of Towbin et al. [15], employing isolated promastigote membrane preparations [3] and using 0.2 /IM pore sized nitrocellulose sheets (Schleicher and Schuell). GP46/M-2 protein was detected using either the monoclonal antibody, M-2 which is specific for the protein of L. amazonensis [16] or polyclonal antisera raised in mice immunized with either wild type vaccinia (negative immune control sera) or recombinant GP46/M-2A using two different vaccinia expression vectors (D. Rodriguez, J. Rodriquez, L. Rivas, K.L.L., M.

153 TABLE 1 Strains and species employed in this study Stock code

Species identification

Designation

Approximate SignaP ~ GP46/M-2 chromosome size

Source b

MHOM/DO/00/450B MHOM/MX/87/Mexl MHOM/MX/87/Mex10 MNYC/BZ/62/M379 MHOM/PA/78/WR227 MHOM/BZ/82/BEL21 MHOM/BR/77/LTB0016 MPRO/BR/76/M4588 MHOM/BR/81/BOS-2 MORY/PA/79/GML3 MHOM/VE/74/PM-H3 MHOM/VE/75/HM76

500 kb 500 kb 500 kb 500 kb 8-900 kb 8 900 kb 8 900 kb 8 900 kb 8-900 kb 600 kb 5-600 kb 8 900 kb

i i i a c e f a j a a a

MHOM/CO/82/CLO64 MHOM/CR/81/734 M H O M / P A / 7 4 / W R 120 MHOM/BR/75/M2903 MHOM/BR/79/LTB014 M H O M / B R / 7 0 / M 1"176 MHOM/BR/75/M4147

ND d e

L. rnexicana complex

450B Mex I Mexl0 Lll c WR227 BEL21 LTB0016 M4588 BOS-2 M6331 PM-H3 c M5903

L. L. L. L. L. L. L. L. L. L. L. L.

mexlcana complex mexlcana mexlcana mexlcana mexlcana mexlcana amazonensis amazonensis amazonensis aristedesi venezuelensis garnhami

L. braziliensis complex CLO64 L. panamensts L. panamensts CR734-81 WRI20 L. panamensts M2903 c L. braziliensis L. braziliensis LTB008 M1176 L. guyanensts M4147 ~ L. guyanensts IM350 L. guyanensts

Other Leishmania species LRC-L38 L. mq]or 252 L. mq/or LV9 c L. donovani LTC-I L. tarentolae Other genera CFC- 1 C. jasciculata LV88 E. monterogeii Peru T. cruzi

Moderate Moderate Moderate Moderate V. Strong V. Strong V. Strong V. Strong V. Strong Strong Moderate V. Strong ND

IUMB/BR/81/IM350

RHO/SU/XX/LRC-L38 MHOM/IR/83/LT252 MHOM/ET/67/L82 LTC-I CFC- 1 LV88 PERU

n k c a b a a m

ND 900 kb 800 kb 900 kb

ND V. Strong Strong Strong

1100 kb

Moderate

~This characterizes the intensity of hybridization. As discussed in the text, these experiments do not determine whether the variability observed arises from sequence divergence or changes in gene copy number. bSources of strains were as follows: (a) Drs. J.J. Shaw and R. Lainson; (b) Dr. P. Marsden; (c) Dr. L. Hendricks; (d) Dr. C.C. Wang; (e) Dr. D. Evans; (f) Dr. M. Hommel; (g) Dr. L. Simpson; (h) Dr. S. Mesnick; (i) Dr. F. Neva; (j) Dr. D. McMahonPratt; (k) Dr. L. Mata: (1) Dr. M. Chance; (m) Dr. J. Arias; (n) Dr. R. Tesh; (o) Dr. L. Schnur. CWorld Health Organization Leishmania reference strains. aND, not determined. e( ) indicates no detectable signal.

Esteban and D.McM.-P. in preparation). The GP46/M-2 polyclonal antisera recognized the native GP46/M-2 of L. amazonensis and previously characterized major proteolytic fragments of the protein [3]. Antigens bound by the monoclonal antibody or the mouse antiGP46/M-2 serum were identified with the use of [125I]radioiodinated rabbit F(ab'2) antimouse immunoglobulin (2 x 105 cpm ml-1; spec. act. 10 fCi/fg). Blots were dried and exposed at - 7 0 ° C for autoradiography.

Results

Chromosomal distribution of the GP46/M-2 gene within the L. mexicana complex. Chromosomes of 12 members of the L. mexicana complex were separated by pulsed field electrophoresis and examined by Southern blotting with a GP46A probe. GP46/M-2 hybridization was observed in all species tested, although 3 types of hybridization pattern were obtained

154

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Fig. 1. Molecular karyotype analysis of GP46/M-2 genes in the L. mexieana species complex. The experimental conditions are as indicated in Materials and Methods. Shown are the autoradiographic results from Southern blot experiments of electrophoretically separated Leishmania chromosomes and a radiolabeled GP46/M-2 probe. The BamHl-Sphl restriction fragment of the L. amazonensLs' GP46A gene was utilized, which contains all but the last 200 bp of the GP46A/M-2 coding region [I 3]. Molecular weight markers (kb) are as indicated.

(Fig. 1; Table 1). In 6 lines (L. mexicana WR227, Bel21: all three L. amazonensis and Leishmania garnhami), strong hybridization was observed to chromosomes of 800 950 kb (Pattern 1), one chromosome in L. mexicana WR227 and L. garnhami and 2 chromosomes in each of the remaining 4 stocks. Hybridization to the sample loading well was also evident. In Leishmania aristedesi somewhat less intense hybridization was observed to a single 600-kb chromosome as well as to the sample well (Pattern 2; Fig. 1). In 4 lines of L. mexicana (450B, MEX1, MEXI0, LI1) and Leishmania venezuelensis, moderate hybridization was observed to a single 500 600 kb chromosome (Pattern 3; Fig. 1). These data

revealed a complex karyotypic distribution of GP46/M-2 within this species complex, although they do not determine whether changes in copy number or sequence divergence are responsible.

Chromosomal distribution of GP46/M-2 genes in other species complexes and genera. Chromosomes from representatives of three other complexes of pathogenic Leishmania ( L. ma/or. L. donovani, L. guyanensis), the lizard parasite Leishmania tarentolae, and the genera Crithidia, Endotrvpanum and Trypanosoma (Trypanosoma cruzi) were separated by pulsed field electrophoresis and examined by Southern blotting with the GP46/M-2 probe (Fig. 2A,B). Examination of the ethidium bromide stained gel (Fig. 2C) and hybridization with a Leishmania ribosomal D N A probe (data not shown) indicated comparable amounts of D N A in all samples. L. ma/or and L. donovani chromosomes of 930 and 850 kb exhibited strong hybridization comparable to that seen in L. amazonensis LTB0016. A 950-kb chromosome in L, tarentolae showed moderate hybridization comparable to that seen with some lines of L. mexicana and L. t'enezuelensis, and a l l00-kb chromosome in Crithidia hybridized weakly (Fig. 2A,B; Fig. 1; Table I). As mentioned earlier, these experiments do not determine whether the variability observed arises from sequence divergence or changes in gene copy number. Finally, faint hybridization to 2 3 chromosomes in both Endoto'panum and T. cruel was also observed, although the hybridization intensity in these species was only slightly above background and may not be significant (Fig. 2B). In support of the hybridization data, GP46/ M-2-related sequences have been recently isolated from molecular recombinant libraries of both L. mq/or (S. Dasgupta, P. Murray, personal communications) and Crithidia (D.McM.-P., unpublished data). Surprisingly, in L. guyanensis, no convincing hybridization was observed, other than a faint nonspecific hybridization to many other chromosomes that was also evident in the

155

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Fig. 2. Molecular karyotype analysis of GP46/M-2 genes in various genera of kinetoplastid flagellates. Southern was performed as described in Fig. 1. The specific strains were: L. amazonensis (LTB0016), L. guyanensis fasciculata (CFC-I), Endotrypanum monterogei (LV89), T. cruzi, L.-major (LT252), L. donovani (LV9) and (M5903). Panels A and B differ only in the length of exposure of the hybridized blot to the film. In Panel C bromide stained agarose gel is shown. Molecular weight markers (kb) are as indicated.

other species (Fig. 2B). This suggested that the GP46/M-2 gene was absent in this species. To confirm this, chromosomes were prepared from other members of the L. braziliensis complex, spanning a wide geographical range and including representative strains of all 3 major recognized species, L. guyanensis, Leishmania panamensis and L. braziliensis (Fig. 3; Table I). A long exposure of a blot of separated chromosomes probed with GP46/ M-2 showed no hybridization above background to any of these species, although massive hybridization was seen to the positive controls L. major and L. amazonensis (Fig. 3, right-hand panel; the faint pattern seen is comparable to that observed with nonspecific control hybridization probes such as pBR322 in other experiments). Examination of the ethidium bromide-stained gel (Fig. 3, lefthand panel) and hybridization with an L. major ribosomal R N A probe showed that comparable amounts of D N A were present on this blot (data not shown). Thus far, we have not been able to provide evidence for the GP46/M-2-related sequences in the L. braziliensis complex.

blot analysis (M4147), C. L. garnhami the ethidium

Organization of GP46/M-2 genes in L. amazonensis, L. major and L. donovani. GP46/M-2 is known to be encoded as a family of nonidentical genes in L. amazonensis [6,7]. We examined the structure of the GP46/M-2 gene family in L. amazonensis LTB0016, which contains two intensely hybridizing chromosomes (Pattern 1), by Southern blotting of total D N A digested with infrequently cutting restriction enzymes lacking sites in the GP46A gene (Fig. 4A). Digestion with SpeI gave two similarly intense fragments of 590 and 440 kb (Fig. 4A), while Dral gave two fragments of 500 and 350 kb; digests with AseI, SspI, and XbaI also yielded two comparably large fragments differing in size by about 150 kb (data not shown). In contrast, NdeI digestion yielded at least 6 fragments, ranging from 8 to l l 0 kb (Fig. 4A). Assuming the absence of comigrating fragments, they total approximately 280 kb. Digestions with more frequently cutting enzymes similarly yielded multiple differently sized fragments, however in no case was a pattern indicative of a single tandemly repeating repetitive array obtained (Fig. 4 and data not shown; see also ref. 6).

156

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Fig. 3. Molecular karyotype analysis of GP46/M-2 genes in the L. braziliensiscomplex• The experimental conditions are those indicated in Fig. 1, except that CHEF electrophoresis was performed using a pulse time orS0 s t\)r 18 h followed by a pulse time of 160 s for 24 h. In the panel on the left is shown the ethidium bromide-stained agarose gel. On the right are shown the autoradiographic results from the Southern blot analyses of the gel. The film was deliberately overexposed to emphasize the lack of specific hybridization to chromosomes of the L. hraziliensiscomplex. Molecular weight markers (kb) are as indicated• The data with the 5 infrequently cutting enzymes provide evidence for 2 clusters o f G P 4 6 / M - 2 genes in L. amazonensis LTB0016. Interestingly, the two G P 4 6 / M - 2 c h r o m o s o m e s differ by a p p r o x i m a t e l y 150 kb, as do the 2 large fragments with the 5 infrequent cutters. This suggests that each G P 4 6 / M - 2 cluster is located on a differently sized c h r o m o s o m e . The simplest explanation is that an expansion o f one G P 4 6 cluster occurred on one c h r o m o some h o m o l o g , in contrast to isolates such as L. mexicana W R 2 2 7 and L l l which possess a single G P 4 6 / M - 2 hybridizing c h r o m o s o m e (Fig. 1). H o m o l o g o u s c h r o m o s o m e s differing in size have been r e p o r t e d in Leishmania [13,17 21] and t r y p a n o s o m e s [22 25]. The organization o f the G P 4 6 / M - 2 genes in L. major and L. donovani was examined by S o u t h e r n blotting with G P 4 6 / M - 2 probes after

digestion with infrequently cutting enzymes. As observed with L. amazonensis, with some enzymes one or two large fragments were obtained, while with others a n u m b e r o f smaller fragments were obtained (Fig. 4B,C). With no e n z y m e was evidence o f a uniform tandemly repeated array obtained (data not shown). These data indicate that as for L. amazonensis, G P 4 6 / M - 2 genes are organized in a cluster o f nonidentical genes in these species.

Western blot analyses ji~r G P46/ M-2 expression in Leishrnania species. The expression o f the G P 4 6 / M - 2 protein in p r o m a s t i g o t e m e m b r a n e preparations was examined by Western blol analyses, using two polyclonal murine antisera directed against the G P 4 6 A protein produced by 2 different vaccinia vectors. Crossreactive molecules were recognized by the polyclonal

157

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B

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C

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Mr

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(kb)

(kb) 40O --

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Fig. 4. Southern blot analysis of O P 4 6 / M - 2 genes in L. mqjor (LT252), L. donovani (LV9), and L. mexicana (M379). C h r o m o s o m a l D N A s were digested with the indicated enzymes as detailed in Materials and Methods and resolved by C H E F electrophoresis for 20 h using a 5 to 50 s ramp at 200 V. Gels w e r e s u b j e c t e d to Southern blot analysis with the HindlII-Sphl fragment of the GP46A gene [6]. Identical results were obtained using the SalI-Hindlll fragment of the GP46A gene (not

shown). Molecular weight markers (kb) are as indicated for each panel.

L. panamensis

L. braziliensis

L. donovani

L. major

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Fig. 5. Shown are the autoradiographic results from Western blot analysis of GP46/M-2 expression in 5 species of Leis'hmania Membrane fractions isolated from L. amazonensis (LTB0016), L. mqjor (LRC-L38), L. donovani (LV9), L. braziliensis (M2903) and L. panamensis (CLO64) were used as the source of antigen as described in Materials and Methods. In the case of L. major, L. donovani and L. amazonensis, the murine antisera employed included: pre-immune/normal mouse sera (lanes 1); antivaccinia (wild type) control sera (lanes 2); anti-GP46/M-2-vaccinia recombinant sera (wild type) (lanes 3); anti-GP46/M-2vaccinia recombinant scra (mutant vector) (lanes 4); monoclonal antibody M-2 (specific for L. amazonensis) (lanes 5). For L. braziliensis and L. panamensis the murine antisera employed included: pre-immune/normal mouse sera (lanes 1); anti-GP46/M2-vaccinia recombinant sera (wild type) (lanes 2); anti-GP46/M-2-vaccinia recombinant sera (mutant vector) (lanes 3); monoclonal antibody M-2 (lanes 4); monoclonal antibody B-21, specific for L. braziliensis and L. panamensis (lanes 5); monoclonal antibody L-l, cross-reactive for all Leishmania species (lanes 5). Molecular weight markers (kDa) are as indicated.

158

GP46/M-2 antisera in L. major and L. donovani. Although positive control monoclonal antibodies (Fig. 5, lanes 5 and 6) gave significant reactions under these experimental conditions with the L. panamensis and L. braziliensis membrane preparations, no crossreactions were observed with the GP46/M-2 polyclonal sera (Fig. 5, lanes 2 and 3). Similar results were obtained in other Western blot experiments employing membrane preparations of L. guyanensis (data not shown). As expected, the L. amazonensis GP46-specific monoclonal antibody M-2 only identified GP46/M-2 in this species and not in either L. ma/or or L. donovani (Fig. 6) [3,16]. Proteolytic degradation products characterized previously were observed in L. amazonensis [3], and it is possible that the smaller 40 kDa band evident in L. donovani and L. major arises in a similar manner. The observation that polyclonal GP46 antisera recognizes proteolytic fragments suggests that even a truncated or partially degraded L. panamensis or L. braziliensis protein could have been detected if present. Similar results were obtained in other experiments employing a rabbit antiserum against native GP46/M-2 isolated from L. amazonensis (data not shown). Thus, no evidence for expression of a GP46/M-2-related protein has been obtained in either L. braziliensis complex promastigotes, the stage in which expression is maximal in other species.

Discussion

We have surveyed the evolutionary distribution of the GP46/M-2 gene family in trypanosomatid protozoa, using a coding region probe and conditions of relaxed hybridization stringency. These studies revealed the clear presence of GP46/M-2-related genes in 3 species complexes of Leishmania pathogenic to humans (L. mexicana, L. tropica, L. donovani), the lizard parasite L. tarentolae, and the monogenetic insect parasite Crithidia jasciculata. It is also possible that sequences related to GP46/M-2 occur in Endotwpanum and Twpanosoma cruzi, although additional studies will be

required to confirm this. In all species of Leishmania GP46/M-2 genes are restricted to one or two chromosomes, containing a cluster with multiple nonidentical genes. Within the L. mexicana species complex, at least three different patterns for the chromosomal distribution of GP46/M-2 sequences were found. Association of these patterns with the species classification of each isolate did not always reveal consistency; for example, members of L. mexicana exhibited either pattern 1 or 3. As both the structure of the GP46/M-2 locus within this complex and the evolutionary relationships amongst the isolates examined become better understood, it may be possible to interpret the evolution of the GP46/ M-2 karyotype in the future. Unlike the other Leishmania tested, GP46/ M-2-related sequences could not be detected in 6 members of the L. braziliensis species complex, isolated from a wide geographical area and including the 3 recognized species. These findings were supported by studies of GP46/M-2 protein expression: GP46/M-2 expression was not found in promastigotes of L. hraziliensis or L. panamensis, while GP46/ M-2 protein was readily detected in 1,. donovani and L. ma/'or in addition to L. amazonensis. These studies suggest that the absence of GP46/M-2 sequences may be a useful marker for members of the L. hraziliensis complex. Phylogenetic distribution of GP46/M-2. Phylogenetic methods were employed to analyze the evolutionary distribution of GP46/M-2 in Leishmania. Organismal [26] and molecular evolutionary comparisons of nuclear DNA small subunit ribosomal R N A sequences indicate that CrithMia is an evolutionary outgroup of all pathogenic Leishmania as well as L. tarentolae (K. Nelson and S.M.B., in preparation). As discussed above, current molecular data suggest that Crithidia and possibly more distant trypanosomatids possess members of the GP46/M-2 gene family. The most parsimonious explanation is that a single change was responsible for the absence of GP46/M-2 sequences in the L. hraziliensis

159

complex, occurring after the separation of this lineage from other Leishmania but prior to speciation within this complex. The simplest model that could account for the lack of GP46/M-2 in the L. braziliensis complex is a chromosomal deletion. Since GP46/M-2 genes are organized as a gene cluster in other Leishmania, this could readily be accomplished in a single genetic event. Chromosomal changes including expansion and contraction of repetitive gene families have been shown or postulated in numerous isolates of Leishmania [13,17 21], although in no instance has complete loss of a gene family been reported. Alternatively, it is possible that GP46/M-2 has undergone rapid evolution in the L. braziliensis complex, sufficiently so that the available antibody and hybridization probes no longer recognize this protein or gene. Although all gene family members would have to undergo accelerated evolution, mechanisms such as concerted evolution [27] could allow all members to evolve in parallel. Although current data do not allow us to choose between the deletion or accelerated divergence models, we currently favor the deletion model. In any event, it is clear that the GP46/M-2 gene family shows a greater variability in evolutionary distribution than any other leishmanial protein antigen characterized thus far. For example, the surface antigen gp63 is present in all Leishmania species complexes and Crithidia [28,29]. This suggests that GP46/M-2 may be associated with features of the parasite life cycle that vary among different species complexes. Unfortunately, the predicted protein sequence of a GP46/M-2 gene from L. amazonensis does not reveal affinity to any known protein that could suggest a potential function [6]. It is known that GP46/M-2 is maximally expressed in the promastigote stage within the sand fly digestive tract (refs. 1, 2 and 30; and D.McM.-P. et al., in preparation). Interestingly, one feature that distinguishes the L. braziliensis complex from other Leishmania is that these promastigotes are primarily found within the hindgut, whereas promastigotes of the other pathogenic species complexes pri-

marily reside within the midgut [26,31]. Conflicting data have been reported with L. tarentolae [31], although one group has reported midgut development in Phlebotomus papatasii [32]. This group also reported midgut development for C. fasciculata, although this organism is normally carried by mosquitoes [33]. These observations raise the possibility that GP46/M-2 may affect parasite growth within the midgut of the fly. In one sense, members of the L. braziliensis complex constitute evolutionarily derived GP46/M-2 'null' mutants. The advent of methods for stable DNA transfection of pathogenic Leishmania [9] will permit exploitation of these 'mutants' in genetic tests of GP46/ M-2 function. We have developed a molecular construct capable of directing GP46/M-2 expression, and succesfully introduced it into L. panamensis, a member of the L. braziliensis species complex [7,34]. The phenotype of these genetically modified parasites during the infectious cycle in the sand fly and macrophages may provide important clues about the biological role of GP46/M-2 in the future.

Acknowledgements The authors would like to thank Ms. Kelledy Manson for excellent technical assistance. This work was supported by grants from the National Institutes of Health AI-23004 (D.McM.-P) and AI-21903 (SMB). Yara Traub-Cseko was supported by a CAPES/ Fulbright Fellowship.

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